Plasmids are small, dispensable chromosomes found in bacteria. Plasmids are medically important since they frequently carry the genes for toxins, antibiotic resistance, and other virulence determinants. Since they are easily transmitted from one bacterium to another, they can also facilitate the rapid dissemination of such virulence determinants. The P1 plasmid addiction operon enhances the retention the of P1 plasmid in E. coli by killing off any cells which fail to retain the plasmid. The operon encodes a toxin that kills plasmid-free cells, and an unstable antidote that prevents killing as long as the plasmid is retained. The andidote, Phd, is a small 73-amino acid protein with a remarkable number of ligands. Phd dimerizes, it binds to DNA (and thus represses transcription of the operen), it binds to the Toxin (and thus neutralizes it) and finally, or alternatively, it is degraded by the ClpXP protease. Since DNA binding and toxin binding can occur simultaneously, these functions must involve different surfaces of Phd. The working hypothesis is that different parts of Phd are involved in different functions. The general aim of the proposed research is to determine what parts of Phd fulfill what functions of Phd. The specific aims are to determine: 1. What parts of Phd neutralize toxin. 2. What parts of Phd repress transcription. 3. What parts of Phd regulate proteolysis. The applicants will use deletion analysis and chimeric analysis to determine which segments of the protein are required for which activities. On a finer scale, the investigators will use site-directed mutagenesis (alanine scan) to determine which amino acid side chains are important for which activities. By testing each mutant for multiple activities, the investigators will be able to determine whether the effects of the mutation are local (affecting a single activity of Phd) or global (affecting multiple activities of Phd). The long-term objective of this research is to understand protein-ligand interactions well enough to recognize recognize ligand binding domains, match proteins to their preferred ligands, design proteins to bind specific ligands and design ligands to bind specific proteins. A superior understanding protein-ligand interactions will assist in the rational design of drugs (ligands) that interact with specific protein targets (agents of disease).